Yamaichi, Y. | Search bioRxiv

bioRxiv2026

Coupling DNA processing to early gene expression drives antibiotic resistance plasmid dissemination

The rapid global spread of antibiotic resistance is mediated by conjugative plasmids, yet how these elements establish immediately after entering new bacterial hosts remains poorly understood. Here we show that plasmids actively coordinate DNA processing with early gene expression to promote their own establishment. Using single-cell measurements of plasmid conversion dynamics, we demonstrate that the F plasmid selectively delays complementary strand synthesis within its leading region, creating a transient single-stranded DNA window that prolongs zygotic induction. This delay is imposed by a single-stranded DNA promoter that forms a structural roadblock to complementary strand DNA synthesis and is resolved by the host helicase UvrD. By extending the single-stranded state of the leading region, plasmids enhance early expression of protective genes that counter host defence responses and promote survival during entry, particularly in non-isogenic hosts. Together, these findings identify early post-entry regulation as a critical determinant of plasmid establishment and reveal how coupling DNA conversion to transient protective gene expression enables conjugative plasmids to overcome host barriers and disseminate antibiotic resistance.

microbiology↗

bioRxiv2022

Real-time visualisation of conjugation reveals the molecular strategy evolved by the conjugative F plasmid to ensure the sequential production of plasmid factors during establishment in the new host cell

DNA conjugation is a contact-dependent horizontal gene transfer mechanism responsible for disseminating drug resistance among bacterial species. Conjugation remains poorly characterised at the cellular scale, particularly regarding the reactions occurring after the plasmid enters the new host cell. Here, we use live-cell microscopy to visualise the intracellular dynamics of conjugation in real time. We reveal that the transfer of the plasmid in single-stranded DNA (ssDNA) form followed by its conversion into double-stranded DNA (dsDNA) are fast and efficient processes that occur with specific timing and subcellular localisation. Notably, the ss-to-dsDNA conversion is the critical step that governs the timing of plasmid-encoded protein production. The leading region that first enters the recipient cell carries single-stranded promoters that allow the early and transient synthesis of leading proteins immediately upon entry of the ssDNA plasmid. The subsequent ss-to-dsDNA conversion turns off leading gene expression and licences the expression of the other plasmid genes under the control of conventional double-stranded promoters. This elegant molecular strategy evolved by the conjugative plasmid allows for the timely production of factors sequentially involved in establishing, maintaining and disseminating the plasmid.

microbiology↗